Classifications

• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
  • C07K14/43504—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from invertebrates
  • C07K14/43595—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from invertebrates from coelenteratae, e.g. medusae
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
  • C12N15/09—Recombinant DNA-technology
  • C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
  • C12N15/1034—Isolating an individual clone by screening libraries
  • C12N15/1055—Protein x Protein interaction, e.g. two hybrid selection
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K2319/00—Fusion polypeptide

Definitions

• the method could be used with a wide variety of proteins and in a wide variety of living cells. Also preferably, the method could be used to determine the interactions between molecules other than proteins.
• a method for determining whether a first protein interacts with a second protein within a living cell comprises providing the first protein complexed to a donor luciferase and the second protein complexed to an acceptor fluorophore within the cell.
• the donor luciferase is capable of luminescence resonance energy transfer to the acceptor fluorophore when the first protein is in proximity to the second protein.
• the complexed first protein and the complexed second protein are allowed to come into proximity to each other within the cell.
• any fluorescence from the acceptor fluorophore is detected. Fluorescence of the acceptor fluorophore resulting from luminescence resonance energy transfer from the donor luciferase to acceptor fluorophore the indicates that the first protein has interacted with the second protein.
• providing the first protein complexed to a donor luciferase and the second protein complexed to an acceptor fluorophore comprises genetically engineering DNA and transferring the genetically engineered DNA to the living cell causing the cell to produce the first protein complexed to a donor luciferase and the second protein complexed to an acceptor fluorophore.
• the cell which is provided with the first protein complexed to a donor luciferase and the cell which is provided with the second protein complexed to an acceptor fluorophore are mammalian cells.
• the donor luciferase provided is Renilla luciferase.
• the acceptor fluorophore provided is an Aequorea green fluorescent protein.
• the detection of acceptor fluorophore fluorescence is performed using spectrofluorometery.
• the present invention includes a method for determining whether a first protein interacts with a second protein in a living cell using luminescent resonance energy transfer (LRET).
• LRET luminescent resonance energy transfer results from the transfer of excited state energy from a donor luciferase to an acceptor fluorophore.
• LRET luminescent resonance energy transfer
• the efficiency of luminescence resonance energy transfer is dependent on the distance separating the donor luciferase and the acceptor fluorophore, among other variables. Generally, significant energy transfers occur only where the donor luciferase and acceptor fluorophore are less than about 80 A of each other.
• the present invention utilizes luminescence resonance energy transfer to determine whether an interaction takes place between a first protein and a second protein in a living cell. This is accomplished by complexing a first protein to the donor luciferase and complexing the second protein to the acceptor fluorophore and placing the complexed first protein and the complexed second protein in the cell under conditions suitable for an interaction between the first protein and the second protein to take place. If the first protein interacts with the second protein, the donor luciferase will come close enough to the acceptor fluorophore for luminescence resonance energy transfer to take place and the acceptor fluorophore will fluoresce.
• this method allows for the detection of interaction between the first protein and the second protein even though the interaction cannot be detected by optical methods such as conventional microscopy.
• the specific labeling of the proteins in living cells can be achieved through genetic engineering methods where the introduction of fluorescent dyes into living cells is very difficult. Further, fluorescent dyes photobleach quickly while light emission of a luciferase such as Renilla luciferase originates from an enzymatic reaction that is relatively stable if substrate and oxygen are supplemented.
• complexing a first protein to the donor luciferase refers to joining the donor luciferase to the first protein in a manner that the donor luciferase and the first protein stay in essentially the same proximity to one another during interaction between the first protein and the second protein.
• complexing a second protein to the acceptor fluorophore refers to joining the acceptor fluorophore to the second protein in a manner that the acceptor fluorophore and the second protein stay in essentially the same proximity to one another during interaction between the first protein and the second protein.
• Such complexing can be done, for example, by genetically engineering the cell to produce a fusion protein containing the donor luciferase and first protein, and the acceptor fluorophore and the second protein.
• the present invention uses Renilla luciferase as the donor luciferase and "humanized" Aequorea green fluorescent protein ('humanized' GFP) as the acceptor fluorophore.
• Renilla luciferase is a 34 kDa enzyme purified from Renilla reniformis. The enzyme catalyzes the oxidative decarboxylation of coelenterazine in the presence of oxygen to produce blue light with an emission wavelength maximum of 471 nm.
• Renilla luciferase was used as the donor luciferase because it requires an exogenous substrate rather than exogenous light for excitation. This, advantageously, eliminates background noise from an exogenous light source and from autofluorescence, and allows easy and accurate quantitative determination of light production.
• 'Humanized' GFP is a 27 kDa protein fluorophore that has an excitation maximum at 480 nm. It has a single amino acid difference from wild-type Aequorea green fluorescent protein. 'Humanized' GFP was chosen as the acceptor fluorophore because its excitation spectrum overlaps with the emission spectra of Renilla luciferase. Additionally, emissions from 'humanized' GFP can be visualized in living cells. Further, 'humanized'
• GFP is expressed well in the mammalian cells transfected with 'humanized' GFP cDNA that were used to demonstrate this method.
• IGFBP 6 insulin-like growth factor binding protein 6
• IGF- II insulin-like growth factor II
• the Renilla luciferase cDNA was fused to IGFBP 6 cDNA and 'humanized' GFP cDNA was fused to IGF-II cDNA.
• Living cells were transfected with the fused cDNAs and the fusion proteins were expressed. Cell extracts were produced and mixed.
• the substrate for the Renilla luciferase moiety of the fused Renilla luciferase-IGFPB 6 protein was added. Finally, fluorescence from the 'humanized' GFP moiety of the fused 'humanized' GFP-IGF-II protein was detected. Demonstration one method according to the present invention will now be described in greater detail.
• IGFBP-6 cDNA SEQ ID NO: l, GenBank accession number M69054, encoded IGFBP-6, SEQ ID NO:2, which was used as the first protein.
• Renilla luciferase cDNA SEQ ID NO:3, GenBank accession number M63501, encoded Renilla luciferase, SEQ ID NO:4, which was used as the donor luciferase.
• Insulin cDNA SEQ ID NO:9, accession number AH002844, encoded insulin, SEQ ID NO: 10.
• Insulin, fused to 'humanized' GFP was used as a control protein because insulin is homologous to IGF-II, but it does not bind to IGFBP-6.
• the cDNA of prepro-IGF-II carried on an EcoRI fragment was cloned into pBluescript KS (+) II vector.
• the insert was sequenced using T7 and T3 primers and confirmed to contain the known cDNA sequence of prepro-IGF-II.
• the 5' end of the IGF-II precursor was connected to the T7 promoter in the pBluescript KS (+) II vector.
• An IGF-II 3' primer was designed to generate a Notice of Allowance restriction site, to remove the D and E domains of prepro-IGF-II, and to maintain the Notice of Allowance fragment of the 'humanized' GFP in frame with the open reading frame of IGF-II.
• the IGF-II fragment was amplified with PCR using the T7 promoter primer and the IGF-II 3' primer.
• the PCR-amplified IGF-II fragment was digested by EcoRI and Not I and cloned into pCDNA3.1 (+) vector (Invitrogen, Carlsbad, CA, US) producing pCDNA-IGF-II.
• the Notice of Allowance fragment of the 'humanized' GFP was inserted into the Not I site of pCDNA-IGF-II producing pC-IGF-II-GFP.
• the cDNA for precursor of insulin which contained a signal peptide the B, C and A domains, was modified in a manner corresponding to the IGF-II fragment, above.
• the 'humanized' GFP cDNA was then linked to the 3' end of the modified insulin cDNA to produce pC-INS-GFP.
• IGFBP 6 cDNA was amplified by PCR from a plasmid named
• Rat-tagged human IGFBP6 Rat-tagged human IGFBP6.
• the stop codon of IGFBP 6 was removed and the open reading frame of IGFBP 6 was in frame with Renilla luciferase cDNA from pCEP4-RUC (Mayerhofer R, Langridge WHR, Cormier MG and Szalay AA. Expression of recombinant Renilla luciferase in trans genie plants results in high levels of light emission. The Plant Journal 1995 ;7; 1031-8).
• the linking of the Renilla luciferase cDNA to the 3' end of modified IGFBP 6 cDNA produced pC-IGFBP 6-RUC.
• COS-7 cells African green monkey kidney cell, American Type Culture Collection CRL 1651
• DMEM Dulbecco's Modified Eagle Medium
• streptomycin 100 mg/ml antibiotic antimycotic solution containing a final concentration of penicillin 100 unit/ml, streptomycin 100 mg/ml and amphotericin B 250 ng/ml (Sigma-Aldrich Co., St. Louis, MO, US) in 5% CO 2 .
• Groups of 1x10° of these cells were plated the day before transfection and were approximately 50% to 60% confluent at the time of transfection. Forty mg of each plasmid fusion DNA were precipitated and resuspended into
• fusion proteins IGF-II-GFP and IGFBP 6-RUC having the expected molecular weights of about 36 kDa and 56 kDa, respectively, were detected using immunoblot analysis. This confirmed the presence of both fusion proteins in the transiently transfected cells.
• cell extracts from these transiently transfected cells were used to carry out a protein binding assay based on energy transfer between the Renilla luciferase and 'humanized' GFP moieties of the fusion proteins.
• the COS cells were washed twice with PBS and harvested using a cell scraper in luciferase assay buffer containing 0.5 M NaCl, 1 mM EDTA and 0.1 M potassium phosphate at a pH 7.5.
• the harvested cells were sonicated 3 times for 10 seconds with an interval of 10 seconds using a Fisher Model 550 Sonic Dismembrator (Fisher Scientific, Pittsburgh, PA, US) to produce cell extracts.
• the cell extracts containing IGF-II-GFP and IGFBP 6-RUC were mixed and 0.1 ⁇ g of coelenterazine was immediately added.
• Spectrofluorometry was performed using a SPEX FluoroMax ® (Instruments S.A., Inc., Edison, NJ). The spectrum showed a single emission peak at 471 nm, which corresponds to the known emission of Renilla luciferase.
• the spectrofluorometry of the cell extracts was carried out at a longer time, but the spectral pattern did not change over time.
• Control cell extract mixtures from cells transfected with pC-INS-GFP and pC-IGFBP 6-RUC were made similarly and their spectra traced.
• the traces showed only one peak at 471 nm, which corresponds to the emission peak of Renilla luciferase.
• the spectral pattern did not change over time.
• protein-protein interactions were also detected by the detection of LRET using corresponding methods in E. coli cells and mammalian cells which were co-transformed.

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• 1999
  • 1999-09-02 AU AU58056/99A patent/AU752675B2/en not_active Ceased
  • 1999-09-02 CA CA002341314A patent/CA2341314A1/en not_active Abandoned
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